1. Field of the Invention (Technical Field)
This invention relates to peptide-based metal ion-labeled compositions for use as pharmaceuticals, and particularly radiopharmaceuticals, for diagnostic imaging and therapeutic applications, and more particularly for in vivo labeling of platelets for localization and detection of thrombosis, and detecting and treatment of other diseases and conditions.
2. Description of the Related Art, Including Information Disclosed under 37 C.F.R. Sections 1.97-1.99 (Background Art)
The use of biologically active peptides, which are peptides which bind to specific cell surface receptors, have received some consideration as radiopharmaceuticals. Canadian Patent Application 2,016,235, Labeled Chemotactic Pepides of Image Focal Sites of Infection or Inflammation, teaches a method of detecting a site of infection or inflammation, and a method for treating such infection or inflammation, by administration of a labeled or therapeutically-conjugated chemotactic peptide. In that application, the chemotactic peptides are chemically conjugated to DTPA and subsequently labeled with In. The utility of DTPA chelates covalently coupled to polypeptides and similar substances is well known in the art. Hnatowich, DJ, U.S. Pat. Nos. 4,479,930 and 4,668,503. Other bifunctional chelates for radiolabeling peptides, polypeptides and proteins are well known in the art. Other biologically active peptides described include those disclosed by Olexa SA, Knight LC and Budzynski AZ, U.S. Pat. No. 4,427,646, Use of Radiolabeled Pepide Derived From Crosslinked Fibrin Locate Thrombi In Vivo, in which iodination is discussed as a means of radiolabeling. In Morgan CA Jr and Anderson DC, U.S. Pat. No. 4,986,979, Imaging Tissue Sites of Inflammation, use of chelates and direct iodination is disclosed. In Tolman GL, U.S. Pat. No. 4,732,864, Trace-Labeled Conjugates of Metallothionein and Target-Seeking Biologically Active Molecules, the use of metallothionein or metallothionein fragments conjugated to a biologically active molecule, including peptides, is disclosed. The previous methods all employ some conjugation means with a bifunctional chelator in order to effectuate labeling with a radionuclide or other medically useful metal ion, such as a paramagnetic contrast agent. The only exception involves radioiodination; the iodine labeling of proteins or peptides containing tyrosine or histidine residues is well known, for example, by the chloramine-T, iodine monochloride, Iodogen or lactoperoxidase methods.
Under homeostatic conditions, platelets circulate as disc shaped cells that do not interact with other circulating blood cells or vascular endothelium (Buchanan MR: Mechanisms of pathogenesis of arterial thrombosis: potential sites of inhibition by therapeutic compounds, Sem Thrombosis and Hemostasis 14(1988) 33-40). The release of adhesive and coagulant agents associated with platelet activation is held in check by high intraplatelet, and possibly vascular endothelium, levels of cAMP.
Upon injury, platelets rapidly attach a) to dysfunctional or detached endothelial cells and b) to the underlying basement membrane and tissues. Differences in platelet response, correlating to the degree of injury, are due in part to differences in the vessel wall composition of the molecules to which the platelets adhere. For example, type I and III collagens, which are typically associated with smooth muscle cells, promote platelet adhesion, aggregation, and release. In contrast, types IV and V collagens, typically associated with the endothelium, facilitate platelet adhesion but do not generally cause platelet activation.
Platelet-mediated thrombosis is a major pathogenetic mechanism in thrombogenesis and reocclusion after successful thrombolytic therapy, and consequently platelets are frequently used as vehicles for localization of thrombi. Additionally, suppression of platelet aggregation is a frequent target for prevention of blood vessel occlusion or reocclusion. There are a number of clinical conditions in which there are platelet accumulations; these include venous thrombosis, arterial thrombosis, left ventricular thrombosis, pulmonary embolism, inflammatory response secondary to myocardial infarction, endocarditis, bypass graft occlusion, aneurysms, prosthetic arterial graft platelet accumulation or occlusion, cerebral embolism or hemorrhage, traumatic injury with hemorrhage, gastrointestinal hemorrhage, and thrombosis secondary to catheters and other implanted devices.
A variety of diagnostic modalities have been used for conditions involving platelet accumulation. These include contrast venography, impedence plethysmography, and .sup.125 I-fibrinogen uptake for venous thromboembolism; .sup.111 In-labeled platelets for a variety of conditions involving platelet accumulation; and, pulmonary angiography, perfusion lung scanning using .sup.99m Tc-human macroaggregated albumin, and ventilationperfusion lung scanning with radioactive gases or aerosols for pulmonary embolism. Each of these modalities presents serious limitations, and has less than desirable efficacy. .sup.111 In-labeled platelets is the only modality which yields a reliable direct measure of platelet accumulation; however, this method suffers serious limitations, including technical difficulties in ex vivo labeling. In addition, since with .sup.111 In-labeled platelets the labeling is performed ex vivo, and the platelets reinjected and allowed to accumulate before imaging, this method does not provide a measure of existing platelet accumulation. Thus, no commonly used method allows for direct detection of existing platelet accumulation within the body.
Peptides containing the adhesive sequence RGD are under active investigation as anti-thrombotic agents (Imura Y, Stassen J-M, Dunting S, Stockmans F, and Collen D: Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in hamster platelet-rich femoral vein thrombosis model, Blood 80(1992) 1247-1253). Knight et al. (Knight LC, Radcliffe R, Kollman M, Dasika V, Wikander R, Mauer AH, Rodwell JD, and Alvarez V: Thrombus imaging with Tc-99 m synthetic peptides reactive with activated platelets. J Nucl Med 31(1990) 757 (abstract)) have reported on the use of .sup.99m Tc-synthetic peptide-metallothionein complexes which bind to the platelet glycoprotein IIb/IIIa complex to image fresh thrombi in jugular veins. However, peptides which target the glycoprotein IIb/IIIa complex are known to adversely affect platelet aggregation, and consequently a radiopharmaceutical based on such an approach would be expected to have severe dose limitations.
In addition to peptides, radiolabeled monoclonal antibodies specific for platelet-related antigens have been studied as diagnostic radiopharmaceuticals. (Shah VO, Zamora PO, Mills SL, Mann PL, and Comp PC: In vitro studies with the platelet-reactive antibody 50H.19 and its fragments. Thrombosis Research 58(1990) 493-504; Som P, Oster ZH, Yamamoto K, Sacker DF, Brill AB, Zamora PO, Newell KD, and Rhodes BA: Radioimmunoimaging of experimental thrombi in dogs using Tc-99 m labeled monoclonal antibody fragments reactive with human platelets. J Nucl Med 27 (1986) 1315-1320).
Laminin is a basement membrane glycoprotein (M.sub.r =900,000) which has various biological activities including promoting cell attachment, growth, and differentiation. A typical laminin molecule consists of three polypeptide chains, A (440 kd), B1 (200 kd), and B2 (220 kd), that are linked by disulfide bonds to form an asymmetric cross-structure. Multiple, distinct adhesive sequences in laminin appear to mediate specific biological functions, and bind to distinct cell surface receptors (Hynes RO: Integrins: versatility, modulation, and signaling in cell adhesion, Cell 69(1992) 11-25; Yamada KM: Adhesive recognition sequences, J Biol Chem 266(1992) 2809-2812).
Integrin-type receptors on platelets (glycoprotein Ib, the glycoprotein IIb/IIIa complex and glycoprotein IV) have been identified as the major adhesion receptors in platelets, but these glycoproteins do not appear to play a role in the interaction of platelets with the intact laminin molecule (Tandon NN, Holland EA, Kralisz U, Kleinman HK, Robey FA, and Jamieson GA: Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets, Biochem J 274(1991) 535-542). However, platelets do bind to laminin peptide fragments via these receptors (Sonnenberg A, Gehlsen KR, Aumailley M, and Timpl R: Isolation of .alpha. 6.beta.1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment, Exp Cell Res 197(1991) 234-244), suggesting that normally these sites in laminin are cryptic for platelets. One non-integrin platelet receptor for laminin is a 67 kDa receptor which binds to laminin-derived peptide sequences containing Tyr-Ile-Gly-SerArg (YIGSR) (SEQ. ID No. 1) (Tandon et al., supra). This platelet receptor appears to play an important role in the interaction of platelets with the intact laminin molecule. Platelet adherence to laminin via this receptor does not in itself result in platelet activation (Ill CR, Engvall E, and Ruoslahti E: Adhesion of platelets to laminin in the absence of activation. J Cell Biol 99(1984) 2140-2145).
Peptides containing the YIGSR (SEQ. ID No. 1) peptide sequence have been proposed as anti-metastatic agents. Yamada Y, Graf JO, Iwamoto Y, Rober F, Kleinman HK, Sasaki M and Martin GR, U.S. Pat. No. 5,092,885, Peptides with Laminin Activity; Schasteen CS, U.S. Pat. No. 5,039,662, Peptide with Anti-Metastatic Activity. These patents involve longer sequences containing the YIGSR peptide sequence, as well as acylated YIGSR peptide sequences.